The human rod opsin gene (RHO) harbors many mutations that cause retinitis pigmentosa (RP). The folded opsin mRNA is a target for mutation-independent hammerhead ribozyme (hhRz) gene therapy for autosomal dominant (ad) RP. We achieved a potent hhRz candidate therapeutic for adRP that could treat all known human RHO mutations. The long-range goal is to translate effective hhRz therapeutics for RHO adRP into human clinical trials. The objective is to conduct a preclinical proof-of-materials test of this hhz agent and further optimized hhRz agents in mouse models of adRP that are humanized for RHO. The central hypothesis is that the reduction of toxic mutant RHO mRNA and protein will reduce photoreceptor stresses and reduce the rate or stop retinal degeneration due to mutant RHO (P347S). The rationale is that reduction of the P347S RHO mRNA and protein by the mutation-independent hhRz must be combined with reconstitution of wild type (WT) RHO expression (also reduced) to avoid compromise of photoreceptor vitality. To test the central hypothesis and accomplish the objectives the Specific Aims are: Aim 1. Use rational and evolutionary approaches to further optimize catalytic activity of our 725 GUC? human RHO hhRz within the current RNA scaffold. Expected results: By rigorously addressing hhRz RNA structure activity relations (SAR) we expect to manifest further potency-optimized lead hhRzs for preclinical trial and identify the optimum agent for clinical translation. Aim 2. Continue a simple preclinical test of the Knockdown hhRz strategy with rAAV packaged gene therapeutic agents by testing for rescue of retinal degeneration in a partially humanized murine model of adRP that expresses a human mutant rod opsin transgene on the mouse WT RHO background, and test toxicity in a fully humanized mouse model expressing only WT human RHO in mouse rod photoreceptors. RHO hhRz genes are delivered by recombinant adeno-associated virus (rAAV). The adRP mouse has a slow outer retinal degeneration (~50% loss in 6 mo.) that offers a temporal window for therapeutic rescue. Expected results: suppression of toxic mutant RHO will rescue photoreceptors and retinal degeneration. The WT human RHO model has no degeneration and allows test of toxicity related to RHO reduction. Expected results: ERG sensitivity shifts and possible demise of WT photoreceptors will depend on levels of RHO reduction. Aim 3. Test the lead hhRz agent and a reconstituting human WT RHO allele for rescue in a humanized adRP model in which human mutant and WT RHO mRNAs are expressed in mouse rod photoreceptors. Expected results: rescue of retinal degeneration in the fully humanized adRP. The plan is innovative with novel rational and directed evolutionary strategies to improve the existing lead hhRz, and novel mouse models humanized for target mRNAs to simulate human clinical trial. Significance- successful rescue in humanized adRP mouse models would provide safety and efficacy outcomes to support clinical translation of this gene therapy approach for human RHO adRP.